The present invention relates to oligonucleotides for detection of Vibrio parahaemolyticus for clinical examination, public hygiene, food evaluation or food poisoning evaluation, and to detection methods for Vibrio parahaemolyticus. 
Vibrio parahaemolyticus is known as a common infectious food poisoning bacteria. Over 95% of Vibrio parahaemolyticus isolated from gastroenteritis patients are Kanagawa phenomenon-positive bacteria exhibiting hemolytic activity in Wagatsuma agar medium, whereas 99% of these bacteria isolated from fish and water are Kanagawa phenomenon-negative bacteria. This had suggested a strong relationship between pathogenic Vibrio parahaemolyticus and the Kanagawa phenomenon, and later investigation revealed that the Kanagawa phenomenon is a phenomenon that occurs due to extracellular release of Vibrio parahaemolyticus thermostable direct hemolysin (TDH). As a result, TDH has come to notice as a pathogenic factor of Vibrio parahaemolyticus. More recently, certain pathogenic strains even among the Kanagawa phenomenon-negative strains have been confirmed to have a base sequence similar to that of TDH, and produce a hemolysin (TDH-related hemolysin: TRH) with partially common antigenicity.
Detection and identification of Vibrio parahaemolyticus has hitherto been complicated and time-consuming as it involves enrichment culturing and isolation culturing followed by determination of the Kanagawa phenomenon. Recently, detection and identification of Vibrio parahaemolyticus has been accomplished by the hybridization method using genetic probes specific to sequences in the TDH or TRH genes, but it has been difficult to obtain sufficient detection sensitivity for food evaluation and the like.
Thus, since identification of Vibrio parahaemolyticus requires complex procedures and prolonged periods, and a rapid detection of trace amounts of Vibrio parahaemolyticus in a sample was difficult to accomplish, a rapid and high sensitive detection method has been desired in fields such as food evaluation. In addition, in order to simplify the examination of interest, development of an automatic examination device has also been desired.
For highly sensitive detection, it is preferable to perform the detection after amplifying a specific sequence in the gene to be detected or identified, or in RNA derived from the gene (hereafter, these genes will collectively be referred to as xe2x80x9ctarget nucleic acidxe2x80x9d).
When the target nucleic acid is DNA, Polymerase chain reaction (PCR) is known as an amplification method thereof. This method accomplishes amplification of a specific sequence in the target DNA by repetition of a cycle of heat denaturation, primer annealing and extension reaction in the presence of a pair of primers homologous and complementary to both ends of the specific sequence, as well as a thermostable DNA polymerase. However, the PCR method requires a complicated procedure involving repetition of rapidly increasing and decreasing the temperature, which prevents its automatization. In addition, amplification of a specific sequence by the PCR method requires oligonucleotides highly specific to the specific sequence, and oligonucleotides with high specificity to the target DNA are also required for highly sensitive detection and identification.
As amplification methods in cases where the target nucleic acid is RNA, there are known the NASBA method and 3SR method, whereby the specific sequence is amplified by the concerted action of reverse transcriptase and RNA polymerase. These methods involve a chain reaction, wherein a promoter sequence-containing primer for a specific sequence in the target RNA, reverse transcriptase, and Ribonuclease H are used to synthesize double-stranded DNA containing the promoter sequence, and this double-stranded DNA is used as a template for RNA polymerase-catalyzed synthesis of RNA containing the specific sequence, while the RNA in turn becomes a template for synthesis of double-stranded DNA containing the promoter sequence. The NASBA method and 3SR method can accomplish nucleic acid amplification at a constant temperature, and are therefore considered to be methods suitable for automation. However, since these amplification methods involve reaction at relatively low temperature (for example, 41xc2x0 C.), the target RNA forms an intramolecular structure which inhibits binding of the primer, and may reduce the reaction efficiency. Consequently, a procedure of heat denaturation of the target RNA prior to the amplification reaction was required to break down the intramolecular structure of the target RNA, thereby to improve the primer binding efficiency. In addition, amplification of the specific sequence by the NASBA method requires an oligonucleotide with high specificity for the specific sequence, and an oligonucleotide with high specificity to the target RNA is also required for highly sensitive detection and identification. Even for RNA detection at low temperature, it is necessary to use an oligonucleotide that can bind to RNA that has formed the aforementioned intramolecular structure.
Therefore, the first object of the present invention is to provide an oligonucleotide that is useful for specific amplification of Vibrio parahaemolyticus thermostable direct hemolysin-related hemolysin genes (trh1 and trh2) or RNA derived from the genes, as well as for their highly sensitive detection and identification.
The second object of the present invention is to provide an oligonucleotide that is useful for specific amplification of Vibrio parahaemolyticus thermostable direct hemolysin gene (tdh2) or RNA derived from this gene, as well as for its highly sensitive detection and identification.
The third object of the present invention to provide a suitable combination of oligonucleotides useful for specific amplification of RNA derived from a Vibrio parahaemolyticus thermostable direct hemolysin-related hemolysin gene (trh1) at relatively low temperature (for example, 41xc2x0 C.), as well as for highly sensitive detection and identification thereof.
The fourth object of the present invention to provide a suitable combination of oligonucleotides useful for specific amplification of RNA derived from a Vibrio parahaemolyticus thermostable direct hemolysin-related hemolysin gene (trh2) at relatively low temperature (for example, 41xc2x0 C.), as well as for highly sensitive detection and identification thereof.
The fifth object of the present invention to provide a suitable combination of oligonucleotides useful for specific amplification of RNA derived from a Vibrio parahaemolyticus thermostable direct hemolysin gene (tdh2) at relatively low temperature (for example, 41xc2x0 C.), as well as for highly sensitive detection and identification thereof.
The invention of an oligonucleotide for detection or amplification of Vibrio parahaemolyticus thermostable direct hemolysin-related hemolysin genes (trh1 and trh2) or RNA derived from these genes, which oligonucleotide is capable of binding specifically to trh1 and trh2 or RNA derived therefrom, and comprises at least 10 contiguous bases of any of the sequences listed as SEQ. ID. Nos. 1 to 11, or an oligonucleotide complementary to said oligonucleotide, which has been accomplished to achieve the first object, relates to an oligonucleotide for detection or amplification of Vibrio parahaemolyticus thermostable direct hemolysin-related hemolysin genes (trh1 and trh2) or RNA derived from these genes, which oligonucleotide is capable of binding specifically to trh1 and trh2 or RNA derived therefrom, and comprises at least 10 contiguous bases of any of the sequences listed as SEQ. ID. Nos. 1 to 11, or an oligonucleotide complementary to said oligonucleotide.
The invention of an oligonucleotide for detection or amplification of a Vibrio parahaemolyticus thermostable direct hemolysin-related hemolysin gene (trh1) or RNA derived from said gene, which oligonucleotide is capable of binding specifically to trh1 or RNA derived therefrom, and comprises at least 10 contiguous bases of any of the sequences listed as SEQ. ID. Nos. 12 to 14, or an oligonucleotide complementary to said oligonucleotide, which has also been accomplished to achieve the aforementioned object, relates to an oligonucleotide for detection or amplification of a Vibrio parahaemolyticus thermostable direct hemolysin-related hemolysin gene (trh1) or RNA derived from said gene, which oligonucleotide is capable of binding specifically to trh1 or RNA derived therefrom, and comprises at least 10 contiguous bases of any of the sequences listed as SEQ. ID. Nos. 12 to 14, or an oligonucleotide complementary to said oligonucleotide.
The invention of an oligonucleotide for detection or amplification of a Vibrio parahaemolyticus thermostable direct hemolysin-related hemolysin gene (trh2) or RNA derived from said gene, which oligonucleotide is capable of binding specifically to trh2 or RNA derived therefrom, and comprises at least 10 contiguous bases of any of the sequences listed as SEQ. ID. Nos. 15 to 17, or an oligonucleotide complementary to said oligonucleotide, which has also been accomplished to achieve the aforementioned object, relates to an oligonucleotide for detection or amplification of a Vibrio parahaemolyticus thermostable direct hemolysin-related hemolysin gene (trh2) or RNA derived from said gene, which oligonucleotide is capable of binding specifically to trh2 or RNA derived therefrom, and comprises at least 10 contiguous bases of any of the sequences listed as SEQ. ID. Nos. 15 to 17, or an oligonucleotide complementary to said oligonucleotide.
The invention also concerns an oligonucleotide primer for a DNA extension reaction, comprising the above-described oligonucleotides. The invention also concerns an oligonucleotide probe a portion of which is modified or labeled with a detectable marker.
The invention of an oligonucleotide for detection or amplification of Vibrio parahaemolyticus thermostable direct hemolysin gene (tdh2) or RNA derived from said gene, which oligonucleotide is capable of binding specifically to tdh2 or RNA derived from said gene, and comprises at least 10 contiguous bases of any of the sequences listed as SEQ. ID. Nos. 18 to 24, or an oligonucleotide complementary to said oligonucleotide, which has been accomplished to achieve the second aforementioned object, relates to an oligonucleotide for detection or amplification of Vibrio parahaemolyticus thermostable direct hemolysin gene (tdh2) or RNA derived from said gene, which oligonucleotide is capable of binding specifically to tdh2 or RNA derived from said gene, and comprises at least 10 contiguous bases of any of the sequences listed as SEQ. ID. Nos. 18 to 24, or an oligonucleotide complementary to said oligonucleotide.
The invention also relates to an oligonucleotide primer for DNA extension reaction, comprising such an oligonucleotide as well as to an oligonucleotide probe comprising such an oligonucleotide, a portion of which is modified or labeled with a detectable marker.
The oligonucleotides of the invention, which have been accomplished to achieve the first and second aforementioned objects, respectively, are oligonucleotides that complementarily bind in a specific manner to intramolecular structure-free regions of the target RNA in the aforementioned RNA amplification, and they are capable of binding specifically to the target RNA without the heat denaturation described above. By providing oligonucleotides that bind to intramolecular structure-free regions of RNA derived from Vibrio parahaemolyticus trh1 and/or trh2 or tdh2 at a relatively low and constant temperature (35-50xc2x0 C., and preferably 41xc2x0 C.), the invention provides oligonucleotides for specific amplification and detection of trh1 and trh2, oligonucleotides for specific amplification and detection of trh1 alone, oligonucleotides for specific amplification and detection of trh2 alone, or oligonucleotides for specific amplification and detection of tdh2. More specifically, the invention encompasses oligonucleotide primers for amplification of target DNA by the PCR method, oligonucleotide primers for amplification of target RNA by the NASBA method, etc., and oligonucleotide probes for detection of target nucleic acid without or after amplification. By use of the oligonucleotides of the invention, a simple, rapid and highly sensitive detection method is provided for food evaluation and food poisoning evaluation.
SEQ. ID. Nos. 1 to 11 represent oligonucleotides for detection or amplification of trh1 and trh2, or RNA derived from these genes. xe2x80x9cRNA derived from these genesxe2x80x9d used herein includes RNA produced by using these genes as templates. The oligonucleotides may be oligonucleotides comprising at least 10 contiguous bases of any of the listed base sequences, or oligonucleotides complementary thereto.
SEQ. ID. Nos. 12 to 14 represent oligonucleotides for detection of trh1 or RNA derived from this gene, and they are useful for amplification or detection of trh1 as distinct from trh2. These oligonucleotides may be oligonucleotides comprising at least 10 contiguous bases of any of the listed base sequences, or oligonucleotides complementary thereto.
SEQ. ID. Nos. 15 to 17 represent oligonucleotides for detection of trh2 or RNA derived from this gene, and they are useful for amplification or detection of trh2 as distinct from trh1. These oligonucleotides may be oligonucleotides comprising at least 10 contiguous bases of any of the listed base sequences, or oligonucleotides complementary thereto.
SEQ. ID. Nos. 18 to 24 represent oligonucleotides for detection or amplification of tdh2, or RNA derived from this gene. xe2x80x9cRNA derived from this genesxe2x80x9d used herein includes RNA produced by using this gene as a template. The oligonucleotides may be oligonucleotides comprising at least 10 contiguous bases of any of the listed base sequences, or oligonucleotides complementary thereto.
One mode of each of the present inventions, to achieve the first or second aforementioned object, is an amplification primer comprising any of the aforementioned oligonucleotides. A nucleic acid amplification reaction carried out using an aforementioned oligonucleotide as the primer allows amplification of the target nucleic acid alone. The amplification method can be PCR method, NASBA method, 3SR method, etc., however, an isothermal nucleic acid amplification method such as NASBA or 3SR method is preferred. Detection of the amplification product by various methods allows detection of Vibrio parahaemolyticus. In this case, one of the aforementioned oligonucleotides other than the oligonucleotide used for amplification may be used as the probe, and fragments of the amplified specific sequence may be confirmed by electrophoresis or the like.
Another mode of each of the present invention to achieve the first or second aforementioned object is a probe which is any of the aforementioned oligonucleotides a portion of which is modified or labeled with a detectable marker. When detecting a target nucleic acid, the oligonucleotide labeled with the detectable marker may be hybridized with the single-stranded target nucleic acid, and the hybridized probe can be detected via the marker. The marker detection may be carried out by a method suitable for the particular marker and, for example, when using an intercalator fluorescent dye for labeling the oligonucleotide, a dye with the property of exhibiting increased fluorescent intensity by intercalation in the double-stranded nucleic acid comprising the target nucleic acid and the oligonucleotide probe may be used in order to allow easy detection of only the hybridized probe without removal of the probe that has not hybridized to the target nucleic acid. When using a common fluorescent dye as the marker, the marker may be detected after removal of the probe that has not hybridized to the target nucleic acid. For the detection, the target nucleic acid in the sample is preferably amplified to a detectable amount by a nucleic acid amplification method such as PCR, NASBA or 3SR method, among which isothermal nucleic acid amplification methods such as the NASBA and 3SR methods are most preferable. When incorporating the nucleotide-labeled probe in the reaction solution during the amplification, it is especially preferable to modify the probe by, for example, adding glycolic acid to the 3xe2x80x2-end so that the probe will not function as a nucleotide primer.
The inventions of a detection method:
(I) employing a RNA amplification process, which comprises the_steps of: forming a cDNA with a RNA-dependent DNA polymerase using a specific sequence of a RNA derived from a Vibrio parahaemolyticus thermostable direct hemolysin-related hemolysin gene (trh1) present in a sample as a template, with a first primer having a sequence complementary to said specific sequence and a second primer having a sequence homologous to said specific sequence, wherein either the first or second primer has a sequence having the RNA polymerase promoter sequence added at its 5xe2x80x2-region, thereby producing a RNA-DNA double-strand; digesting the KNA of said RNA-DNA double-strand with Ribonuclease H to form a single-stranded DNA; and then forming a double-stranded DNA that includes a promoter sequence allowing transcription of said KNA sequence or a RNA comprising a sequence complementary to said RNA sequence with a DNA-dependent DNA polymerase using said single-stranded DNA as a template, said double-stranded DNA produces a RNA transcription product in the presence of a RNA polymerase, and said RNA transcription product is subsequently used as the template for the single-stranded DNA production with said RNA-dependent DNA polymerase; characterized in that the_oligonucleotide of SEQ. ID. No.25 is used as the first primer and the oligonucleotide of SEQ. ID. No.26 is used as the second primer;
(II) employing a RNA amplification process, which comprises the steps of: forming a cDNA with a RNA-dependent DNA polymerase using a specific sequence of a RNA derived from a Vibrio parahaemolyticus thermostable direct hemolysin-related hemolysin gene (trh2) present in a sample as a template, with a first primer having a sequence complementary to said specific sequence and a second primer having a sequence homologous to said specific sequence, wherein either the first or second primer has a sequence_having the RNA polymerase promoter sequence added at its 5xe2x80x2-region, thereby producing a RNA-DNA double-strand; digesting the RNA of said RNA-DNA double-strand with Ribonuclease H to form a single-stranded DNA; and then forming a double-stranded DNA that includes a promoter sequence allowing transcription of said RNA sequence or a RNA comprising a sequence complementary to said RNA sequence with a DNA-dependent DNA polymerase using said single-stranded DNA as a template, said double-stranded DNA produces a_RNA transcription product in the presence of a RNA polymerase, and said RNA transcription product is subsequently used as the template for the single-stranded DNA production with said RNA-dependent DNA polymerase; characterized in that the oligonucleotide of SEQ. ID. No.33 is used as the first primer and the oligonucleotide of SEQ. ID. No.34 is used as the second primer; or
(III) employing a RNA amplification process, which comprises the steps of: forming a cDNA with a RNA-dependent DNA polymerase using a specific sequence of a RNA derived from a Vibrio parahaemolyticus thermostable direct hemolysin gene (tdh) present in a sample as a template, with a first primer having a sequence_complementary to said specific sequence and a second primer having a sequence homologous to said specific sequence, wherein either the first or second primer has a sequence having the RNA polymerase promoter sequence added at its 5xe2x80x2-region, thereby producing a RNADNA double-strand; digesting the RNA of said RNA-DNA double-strand with Ribonuclease H to form a single-stranded DNA; and then forming a double-stranded DNA that includes a promoter sequence allowing transcription of said RNA sequence or a RNA comprising a sequence complementary to said RNA sequence with a DNA-dependent DNA polymerase using said single-stranded DNA as a template, said double-stranded DNA produces a RNA transcription product in the presence of a RNA polymerase, and said RNA_transcription product is subsequently used as the template for the single-stranded DNA production with said RNA-dependent DNA polymerase; characterized in that the oligonucleotide of SEQ. ID. No.39 is used as the first primer and the oligonucleotide of SEQ. ID. No.40 is used as the second primer, or the oligonucleotide of SEQ. ID. No.39 is used as the first primer and the oligonucleotide of SEQ. ID. No.41 is used as the second primer, or the oligonucleotide of SEQ. ID. No.42 is used as the first primer and the oligonucleotide of SEQ. ID. No.41 is used as the second primer; which have been accomplished to achieve the third, fourth and fifth aforementioned objects, respectively, relate to a detection method employing a RNA amplification process, which comprises the steps of: forming a cDNA with a RNA-dependent DNA polymerase using a specific sequence of a RNA derived from trh1, trh2 or tdh2 present in a sample as a template, with a first primer having a sequence complementary to said specific sequence and a second primer having a sequence homologous to said specific sequence, wherein either the first or second primer has a sequence having the RNA polymerase promoter sequence added at its 5xe2x80x2-region, thereby producing a RNA-DNA double-strand; digesting the RNA of said RNA-DNA double-strand with Ribonuclease H to form a single-stranded DNA; and then forming a double-stranded DNA that includes a promoter sequence allowing transcription of said RNA sequence or a RNA comprising a sequence complementary to said RNA sequence with a DNA-dependent DNA polymerase using said single-stranded DNA as a template, said double-stranded DNA produces a RNA transcription product in the presence of a RNA polymerase, and said RNA transcription product is subsequently used as the template for the single-stranded DNA production with said RNA-dependent DNA polymerase; characterized in that, as for trh1, the oligonucleotide of SEQ. ID. No.25 is used as the first primer and the oligonucleotide of SEQ. ID. No.26 is used as the second primer, as for trh2, the oligonucleotide of SEQ. ID. No.33,is used as the first primer and the oligonucleotide of SEQ. ID. No.34 is used as the second primer, as for tdh2, the oligonucleotide of SEQ. ID. No.39 is used as the first primer and the oligonucleotide of SEQ. ID. No.40 is used as the second primer, or the oligonucleotide of SEQ. ID. No.39 is used as the first primer and the oligonucleotide of SEQ. ID. No.41 is used as the second primer, or the oligonucleotide of SEQ. ID. No.42 is used as the first primer and the oligonucleotide of SEQ. ID. No.41 is used as the second primer.
The invention an embodiment of such method (I), characterized in that said first primer is an oligonucleotide comprising at least 10 contiguous bases of the sequence of SEQ. ID. No.25, has been accomplished to achieve the third aforementioned object. The invention also relates to the embodiment of such method (I) characterized in that the second primer is an oligonucleotide comprising at least 10 contiguous bases of the sequence of SEQ. ID. No.26. The invention of a detection method for a Vibrio parahaemolyticus thermostable direct hemolysin-related hemolysin gene, which comprises the steps of:_conducting the RNA amplification process in the presence of an oligonucleotide probe labeled with an intercalator fluorescent dye, wherein the sequence of said probe is complementary to at least a portion of said RNA transcription product, and complementary binding of said probe to said RNA transcription product results in a change of the fluorescent property relative to that of a situation where a complex formation is absent; and then measuring the fluorescence intensity of the reaction solution_relates to an embodiment of such method (I) and is a detection method for a Vibrio parahaemolyticus thermostable direct hemolysin-related hemolysin gene, which comprises the steps of: conducting said RNA amplification process in the presence of an oligonucleotide probe labeled with an intercalator fluorescent dye, wherein the sequence of said probe is complementary to at least a portion of said RNA transcription product, and complementary binding of said probe to said RNA transcription product results in a change of the fluorescent property relative to that of a situation where a complex formation is absent; and then measuring the fluorescence intensity of the reaction solution.
The invention also concerns an embodiment of the detection method (II), characterized in that said first primer is an oligonucleotide comprising at least 10 contiguous bases of the sequence of SEQ. ID. No.33., has been accomplished to achieve the fourth aforementioned object. The invention also relates to the embodiment of such detection method (II) characterized in that the second primer is an oligonucleotide comprising at least 10 contiguous bases of the sequence of SEQ. ID. No.34. The invention also relates to the embodiment of such detection method (II) which comprises the steps of: conducting said RNA amplification process in the presence of an oligonucleotide probe labeled with an intercalator fluorescent dye, wherein the sequence of said probe is complementary to at least a portion of said RNA transcription product, and complementary binding of said probe to said RNA transcription product results in a change of the fluorescent property relative to that of a situation where a complex formation is absent; and then measuring the fluorescence intensity of the reaction solution.
The embodiment of such detection method (III) which has been accomplished to achieve the fifth aforementioned object, is characterized in that the first primer is an oligonucleotide comprising at least 10 contiguous bases of the sequence of SEQ. ID. No.39 or SEQ. ID. No.42. The invention also relates to an embodiment of such detection method (III) characterized in that the second primer is an oligonucleotide comprising at least 10 contiguous bases of the sequence of SEQ. ID. No.40 or SEQ. ID. No.41. The invention also relates to an embodiment of such detection method (III) which comprises the steps of: conducting said KNA amplification process in the presence of an oligonucleotide probe labeled with an intercalator fluorescent dye, wherein the sequence of said probe is complementary to at least a portion of said RNA transcription product, and complementary binding of said probe to said RNA transcription product results in a change of the fluorescent property relative to that of a situation where a complex formation is absent; and then measuring the fluorescence intensity of the reaction solution.
By providing a combination of oligonucleotides for detection of RNA derived from Vibrio parahaemolyticus trh1, trh2 or tdh2, at a relatively low and constant temperature (35-50xc2x0 C., and preferably 41xc2x0 C.), i.e., providing a combination of an oligonucleotide primer for amplification of trh1-derived RNA and an oligonucleotide probe for detection thereof, the inventions which has been accomplished to achieve the third, fourth and fifth objects, respectively, provide a detection method and detection kit for simple, rapid and highly sensitive detection of the Vibrio parahaemolyticus thermostable direct hemolysin-related hemolysin gene (trh1 or trh2) or Vibrio parahaemolyticus thermostable direct hemolysin gene (tdh2), for food evaluation and food poisoning evaluation.
According to one mode of each of the present inventions to achieve the third, fourth or fifth aforementioned object, the first primer (a sequence complementary to the 3xe2x80x2-end region of a specific sequence of the target RNA) complementarily binds to a specific sequence of RNA derived from the Vibrio parahaemolyticus thermostable direct hemolysin-related hemolysin gene (trh1 or trh2) or the Vibrio parahaemolyticus thermostable direct hemolysin gene (tdh2) in a sample as the template, and cDNA is produced by extension reaction with RNA-dependent DNA polymerase to form a RNA-DNA double-strand, after which the RNA of the RNA-DNA double-strand is digested with Ribonuclease H to produce a single-stranded DNA. Next, a second primer (a sequence homologous to the 5xe2x80x2-end region of the target RNA, and includes the RNA polymerase promoter sequence added at its 5xe2x80x2-end) complementarily binds to the single-stranded DNA, to produce a double-stranded DNA having a promoter sequence allowing transcription of RNA comprising a sequence homologous to the target RNA sequence, using DNA-dependent DNA polymerase. The double-stranded DNA is then used for amplification of the RNA transcription product comprising the sequence homologous to the target RNA sequence in the presence of RNA polymerase. The present invention is characterized in that, as for trh1, the oligonucleotide of SEQ. ID. No.25 is used as the first primer and the oligonucleotide of SEQ. ID. No.26 is used as the second primer, as for trh2, the oligonucleotide of SEQ. ID. No.33 is used as the first primer and the oligonucleotide of SEQ. ID. No.34 is used as the second primer, as for tdh2, the oligonucleotide of SEQ. ID. No.39 is used as the first primer and the oligonucleotide of SEQ. ID. No.40 is used as the second primer, or the oligonucleotide of SEQ. ID. No.39 is used as the first primer and the oligonucleotide of SEQ. ID. No.41 is used as the second primer, or the oligonucleotide of SEQ. ID. No.42 is used as the first primer and the oligonucleotide of SEQ. ID. No.41 is used as the second primer. For tdh2, it is particularly preferable to use the oligonucleotide of SEQ. ID. No.42 as the first primer and the oligonucleotide of SEQ. ID. No.41 as the second primer.
The first and second primers may be the full-length base sequences of SEQ. ID. Nos.25, 33, 39, 42 and SEQ. ID. Nos.26, 34, 40, 41, respectively, however, combinations of oligonucleotides comprising at least 10 contiguous bases of these sequences may also be used.
According to this mode of each of the present inventions, to achieve the third, fourth or fifth aforementioned object, the target RNA must be cleaved at the 5xe2x80x2-end of the specific sequence. The method of cleaving the target RNA is preferably a method in which an oligonucleotide (cleaving oligonucleotide) with a sequence complementary to a region overlapping and adjacent to the 5xe2x80x2-end of the specific sequence is added, thereby cleaving the target RNA with Ribonuclease H or the like. The 3xe2x80x2-end of the cleaving oligonucleotide is preferably treated by amination, for example, to prevent it from functioning as an oligonucleotide primer.
According to this mode of each of the present inventions, to achieve the third, fourth or fifth aforementioned objects, the amplification process is preferably carried out in the presence of an oligonucleotide probe labeled with an intercalator fluorescent dye having a sequence complementary to at least a portion of the RNA transcription product. Complementary binding of the probe to the RNA transcription product results in a change of the fluorescent property compared to a situation where the complex formation is absent, so that the fluorescence intensity of the reaction solution may be measured. When a labeled oligonucleotide probe is incorporated during the amplification process, it is particularly preferable to modify the probe by, for example, addition of glycolic acid to the 3xe2x80x2-end, to prevent it from functioning as a primer in the extension reaction.
For trh1, the oligonucleotide probe used may be an oligonucleotide comprising at least 10 contiguous bases of the sequences listed as SEQ. ID. Nos.30 to 32.
For trh2, the oligonucleotide probe used may be an oligonucleotide comprising at least 10 contiguous bases of the sequences listed as SEQ. ID. No.38.
For tdh2, the oligonucleotide probe used may be an oligonucleotide comprising at least 10 contiguous bases of SEQ. ID. No.39, when a combination of oligonucleotides comprising at least 10 contiguous bases of SEQ. ID. No.41 and SEQ. ID. No.42 are used for the RNA amplification process.
According to another mode of each of the present inventions, to achieve the third, fourth or fifth aforementioned object, the first primer (a sequence complementary to the target RNA, and including the RNA polymerase promoter sequence added at the 5xe2x80x2-region) complementarily binds to a specific sequence of RNA derived from trh1, trh2 or tdh2 in a sample as the template, and cDNA is produced by extension reaction with RNA-dependent DNA polymerase to form a RNA-DNA double-strand, after which the RNA of the RNA-DNA double-strand is digested with Ribonuclease H to produce single-stranded DNA. Next, the second primer (a sequence homologous to the target RNA) complementarily binds to the single-stranded DNA, to produce double-stranded DNA having a promoter allowing transcription of RNA comprising a sequence complementary to the target RNA sequence, using DNA-dependent DNA polymerase. The double-stranded DNA is then used to produce a RNA transcription product comprising a sequence complementary to the target RNA, in the presence of RNA polymerase. The second primer complementarily binds to this RNA transcription product (the sequence complementary to the target RNA), and cDNA is produced with RNA-dependent DNA polymerase to form a double-strand of RNA-DNA. Next, the RNA of the RNA-DNA double-strand is digested with Ribonuclease H to produce single-stranded DNA, and the first primer complementarily binds to this single-stranded DNA to produce double-stranded DNA having a promoter allowing transcription of RNA comprising a sequence complementary to the target RNA sequence, using DNA-dependent DNA polymerase. The double-stranded DNA is then used for amplification of the RNA transcription product comprising a sequence complementary to the target RNA sequence in the presence of RNA polymerase. The present invention is characterized in that, as for trh1, the oligonucleotide of SEQ. ID. No.25 is used as the first primer and the oligonucleotide of SEQ. ID. No.26 is used as the second primer, as for trh2, the oligonucleotide of SEQ. ID. No.33 is used as the first primer and the oligonucleotide of SEQ. ID. No.34 is used as the second primer, as for tdh2, the oligonucleotide of SEQ. ID. No.39 is used as the first primer and the oligonucleotide of SEQ. ID. No.40 is used as the second primer, or the oligonucleotide of SEQ. ID. No.39 is used as the first primer and the oligonucleotide of SEQ. ID. No.41 is used as the second primer, or the oligonucleotide of SEQ. ID. No.42 is used as the first primer and the oligonucleotide of SEQ. ID. No.41 is used as the second primer. For tdh2, it is particularly preferable to use the oligonucleotide of SEQ. ID. No.42 as the first primer and the oligonucleotide of SEQ. ID. No.41 as the second primer.
The first and second primers may be the full-length base sequences of SEQ. ID. Nos.25, 33, 39, 42 and SEQ. ID. Nos.26, 34, 40, 41, respectively, however, combinations of oligonucleotides comprising at least 10 contiguous bases of these sequences may also be used.
According to this mode of each of the present inventions, to achieve the third, fourth or fifth aforementioned object, the amplification process is preferably carried out in the presence of an oligonucleotide probe labeled with an intercalator fluorescent dye having a sequence complementary to at least a portion of the RNA transcription product. Complementary binding of the probe to the RNA transcription product produces a change in the fluorescent property relative to that of a situation where the complex formation is absent, so that the fluorescence intensity of the reaction solution may be measured. When a labeled oligonucleotide probe is incorporated during the amplification process, it is particularly preferable to modify the probe by, for example, addition of glycolic acid to the 3xe2x80x2-end, to prevent it from functioning as a primer in the extension reaction.
For trh1, the oligonucleotide probe used may be a sequence complementary to an oligonucleotide comprising at least 10 contiguous bases of the sequences listed as SEQ. ID. Nos.30 to 32.
For trh2, the oligonucleotide probe used may be a sequence complementary to an oligonucleotide comprising at least 10 contiguous bases of the sequences listed as SEQ. ID. No.38.
For tdh2, the oligonucleotide probe used may be a sequence complementary to an oligonucleotide comprising at least 10 contiguous bases of SEQ. ID. No.39, when a combination of oligonucleotides comprising at least 10 contiguous bases of SEQ. ID. No.41 and SEQ. ID. No.42 are used for the RNA amplification process.